System and apparatus for point-of-care diagnostics

ABSTRACT

A system comprised of an apparatus and a test device is described. The test device and the apparatus are designed to interact to determine the presence or absence of an analyte of interest in a sample placed on the test device.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/932,769, filed Nov. 4, 2015, now allowed, which is a continuation ofU.S. application Ser. No. 13/783,019, filed Mar. 1, 2013, now U.S. Pat.No. 9,207,181, which claims the benefit of U.S. Provisional ApplicationNo. 61/666,689, filed Jun. 29, 2012; U.S. Provisional Application No.61/636,105, filed Apr. 20, 2012; and U.S. Provisional Application No.61/605,694, filed Mar. 1, 2012. Each of the aforementioned prioritydocuments is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The subject matter described herein relates to a system and an apparatusfor analysis of a sample to aid in medical diagnosis or detection of thepresence or absence of an analyte in the sample.

BACKGROUND

Diagnostic immunoassays are typically reliant on a color change or theproduction of color to reveal a result that is often visible by thehuman eye. As a result of the human perception and judgment involved,there is significant variance among those interpreting such test resultsas to whether a color change or other measurable signal has occurred,particularly if the signal is close to a threshold value. There can be,therefore, subjectivity involved in interpreting whether immunoassayresults are positive or negative. Moreover, for detection of someanalytes present in small quantities, a sufficient color change or colorproduction is not possible for detection by the human eye. Accordingly,there remains a need in the art for an apparatus and a system thatobjectively analyses a signal from an immunoassay test device, to reducethe error associated with interpreting the result, and to improve thesensitivity of the device by providing an apparatus that can detect asmall or weak signal imperceptible to the human eye.

BRIEF SUMMARY

The following aspects and embodiments thereof described and illustratedbelow are meant to be exemplary and illustrative, not limiting in scope.

In one aspect, an apparatus for detection of a signal from a test deviceindicative of the presence or absence of an analyte in a sample isprovided.

In another aspect, a system comprising an apparatus and a lateral flowimmunoassay test device is provided. The apparatus is comprised of ahousing comprising a drawer movable between an open position and aclosed position in which the drawer is contained within the housing; anoptional bar code scanner positioned in the housing for reading anencoded label on a test assay to be inserted into the apparatus; acarriage movably mounted in the housing, the carriage comprising asource of excitation light and a photodetector for detecting energyemitted; drive electronics to move the carriage sequentially from afirst position to a final position, and a plurality of positions therebetween, wherein the carriage has a dwell time at each of said pluralityof positions between the first and final positions; and a processor forcontrol of the drive electronics and carriage and for processing datadetected by the photodetector.

The test device of the system is comprised of a test strip and anoptional bar code label; a label pad on the test strip, the label padcomprised of microparticles comprised of a fluorescing lanthanidecompound and an antibody with binding specificity of an analyte ofinterest; and a plurality of lines on the test strip, positioneddownstream from the label pad, the plurality of lines comprising atleast a reference line or a control line and an analyte-specific testline. Upon insertion of the test device into the drawer of the apparatusand moving the drawer into its closed position, (i) the bar codescanner, if present, obtains information from the bar code label on thetest device housing, if present, regarding the analyte of interest to bedetected. If the bar code scanner and/or bar code label is/are notpresent, the information is provided by a user, an external bar codescanner, or other mechanism. Then, based on the analyte of interest tobe detected, the processor selects an analyte-specific measurementprotocol wherein the carriage is moved from its first to final position,and at each position the photodetector detects light emitted from thetest strip during an illumination period when the source of excitationlight is on and during a dark period with the source of excitation lightis not powered on. A one-dimensional data array is generated where thedifference between light emitted in the illumination period and thelight emitted in the dark period at each carriage position is generatedby the processor, wherein data in the data array corresponding to thereference line or the control line is used to identify the location ofdata in the array that corresponds to the analyte-specific test line.

In one embodiment, the test device is a lateral flow immunoassay. Inanother embodiment, the test strip is in a housing member.

In one embodiment, the drawer comprises at least one arm for positioningthe test device (e.g., lateral flow immunoassay) in a predefinedposition for interaction with the movable carriage.

In another embodiment, the source of excitation light is a lightemitting diode. For example, the light emitting diode can emits light atabout 365 nm. In another embodiment, the light emitting diode isprovided with at least about 4 mW.

In yet another embodiment, the apparatus further comprises a socket forinsertion of a memory device. In one embodiment, the memory device has aread only capability or a read-write capability.

In yet another embodiment, the apparatus further comprises a port forconnection with an external instrument. In still another embodiment, theexternal instrument is selected from a computer, a storage device, a barcode scanner, and a laboratory instrument.

In one embodiment, an internal bar code scanner is present in theapparatus, and the internal bar code scanner is comprised of a lightsource, a lens and a light sensor that translates optical impulses intoelectrical impulses, and wherein one or more mirrors are positioned toachieve interaction of light from the bar code scanner light source anda bar code label on a test device inserted into the apparatus.

In still another embodiment, the plurality of lines on the test deviceis comprised of, in an upstream to downstream direction with respect toflow of fluid on the test device, a negative control line, ananalyte-specific test one, and a reference line. In one embodiment, thereference line comprises antibodies for non-specific binding toimmunoglobulins present in a sample, and based on its fluorescent signalcommunicates to the processor whether sufficient sample has reached thereference line and provides a positional reference for determining theposition of the analyte-specific test line.

In yet another embodiment, the reference line is dimensionally widerthan the analyte-specific test line.

In one embodiment, a first derivative of the data array is calculated,and a minimum peak and a maximum peak corresponding to the referenceline is used to determine a cutoff value for analysis of datacorresponding to the analyte specific test line.

In another embodiment, the plurality of lines includes a negativecontrol line comprised of antibodies with non-specific binding toimmunoglobulins in a sample.

In another embodiment, a first derivative of the data array iscalculated, and a minimum peak and a maximum peak corresponding to thenegative control line is used to determine a cutoff value for analysisof data corresponding to the analyte specific test line.

In still another embodiment, the fluorescing lanthanide compound iseuropium. In one embodiment, the microparticles are comprised of aeuropium core with a polystyrene exterior.

In yet another embodiment, a splash shield is positioned between theimmunoassay test device and the carriage to protect the photodetectorand/or source of excitation light from sample on the immunoassay testdevice.

In still another embodiment, a sample placed on the test device flows inan upstream to a downstream direction, from a sample pad downstream tothe region comprising a plurality of line, and wherein the movablecarriage scans the test device in a downstream to upstream direction.

In another aspect, a kit, comprising a system as described herein and acalibration cassette comprised of at least two lines that fluoresce uponexcitation with the light source is provided.

In one embodiment, the at least two lines that fluoresce are comprisedof a fluorescing compound deposited on a biaxially-oriented polyester.

In another embodiment, the kit additionally comprises a memory device,such as an SD card, with information encoded or stored thereon.

In another aspect, a method for detecting the presence or absence of ananalyte in a sample is provided. The method comprises applying a sampleto a test device (also referred to generally as a test strip),exemplified by a lateral flow immunoassay, that comprises a plurality oflines comprised of a control line or a reference line and ananalyte-specific test line, wherein each of the line in the pluralitycomprise a label that fluoresces or luminesces upon illumination, andthe analyte-specific test line comprises an antibody specific for ananalyte of interest in the sample, the antibody associated with thedetectable label; inserting the test device into a receiving member inan apparatus comprising a movable optics module, the optic modulecomprised of an illumination source and a photodetector; moving theoptics module incrementally in a downstream to upstream direction withrespect to fluid flow on the test device, where the optics module pausesfor a dwell time at each incremental position, and at each position theillumination source is turned on and then turned off, and thephotodetector detects a signal (e.g., light) emitted by the test devicewhen the illumination source is on and when the illumination source isoff; and processing the emitted light data by generating aone-dimensional data array of the difference in emitted light when theillumination source is on and when the illumination source is off ateach incremental position, and calculating a first derivative of theone-dimensional array to generate a derivative data set, wherein thefirst maximum peak in the derivative data set corresponds to data fromthe reference line or the control line, and is used to identify thelocation of data in the derivative data set for the analyte-specifictest line.

In one embodiment, the analyte of interest is an infectious analyte.Exemplary infectious analytes include a virus or a bacteria, such asinfluenza A or influenza B.

In another embodiment, the test device comprises an analyte-specifictest line for influenza A and an analyte-specific test line forinfluenza B.

In another aspect, an apparatus comprising the following elements isprovided: a housing comprising a drawer movable between an open positionand a closed position where the drawer is contained within the housing;an optional bar code scanner positioned in the housing for reading anencoded label on a test assay (or test device) to be inserted into theapparatus; a carriage movably mounted in the housing, said carriagecomprising a source of excitation light and a photodetector fordetecting energy emitted; drive electronics to move the carriagesequentially from a first position to a final position, and a pluralityof positions there between, wherein the carriage has a dwell time ateach of said plurality of positions between the first and finalpositions; a processor for control of the apparatus, wherein theprocessor comprises a software program that processes data obtained fromthe photodetector by generating a data array comprised of emitted signalat each incremental position between the first and final positions,taking the first derivative of the data array to form a derivative dataset, wherein a first maximal value in the derivative data setcorresponds to a maximum signal from a reference line or a control lineon the test device, and the position in the derivate data array of thefirst maximal value determines the position of data from theanalyte-specific test line.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by reference to thedrawings and by study of the following descriptions.

Additional embodiments of the present systems, apparatus and methodswill be apparent from the following description, drawings, examples, andclaims. As can be appreciated from the foregoing and followingdescription, each and every feature described herein, and each and everycombination of two or more of such features, is included within thescope of the present disclosure provided that the features included insuch a combination are not mutually inconsistent. In addition, anyfeature or combination of features may be specifically excluded from anyembodiment of the system, apparatus or method. Additional aspects andadvantages of the present systems and apparatus are set forth in thefollowing description and claims, particularly when considered inconjunction with the accompanying examples and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are front perspective (FIG. 1A) and a back view (FIG. 1B) ofan exemplary apparatus;

FIGS. 2A-2B are views of an exemplary apparatus showing a side view ofthe apparatus with the drawer in an open position (FIG. 2A) and a topperspective view of the drawer in an open position with a lateral flowimmunoassay test device inserted into the drawer (FIG. 2B);

FIGS. 3A-3B are close-up views of the drawer in the apparatus in an openposition (FIG. 3A) and in a closed position (FIG. 3B), with a testdevice positioned in the drawer;

FIG. 4 is a schematic of the optics system within the apparatus;

FIG. 5 is a view of an exemplary apparatus with an optional bar codescanner device attached;

FIG. 6 is a view of a calibration cassette;

FIG. 7 is an illustration of the software architecture of an apparatus;

FIG. 8 is an illustration of an embodiment of a test device, exemplifiedby a lateral flow immunoassay;

FIGS. 9A-9B are illustrations of a test device wherein a test strip isenclosed in an optional housing sized for insertion into a drawer of anapparatus;

FIG. 10 is a top view of an exemplary test device and the arrangement ofits structural and immunochemical features for interaction with theapparatus;

FIG. 11 shows the sequence of events in one embodiment of a measurementprocedure where an apparatus as described herein interacts with a testdevice;

FIGS. 12A-12B show the sequence of events in another embodiment of ameasurement procedure where an apparatus as described herein interactswith a test device;

FIGS. 13A-13C correspond to a top view of a test device (FIG. 13A), across-sectional view of the test window region of the test device (FIG.13B), and an exploded view of a portion of a test device showing anembodiment of the arrangement of test lines, reference lines and controllines on a test device (FIG. 13C); and

FIGS. 14A-14C are graphs showing an exemplary data set from an opticalscan of a test device for detection of influenza A and influenza B,where the data is shown in arbitrary RLU as a function of position ofthe optics module (FIG. 14A), and the signal peak for the referencecontrol line is presented as the first derivative to illustratefunctioning of the algorithm to determine whether a peak is a maximumand not a minimum (FIG. 14B) and to determine peak height (FIG. 14C).

DETAILED DESCRIPTION I. Definitions

Various aspects now will be described more fully hereinafter. Suchaspects may, however, be embodied in many different forms and should notbe construed as limited to the embodiments set forth herein; rather,these embodiments are provided so that this disclosure will be thoroughand complete, and will fully convey its scope to those skilled in theart.

Where a range of values is provided, it is intended that eachintervening value between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the disclosure. For example, if a range of 1 μm to 8μm is stated, it is intended that 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, and 7 μmare also explicitly disclosed, as well as the range of values greaterthan or equal to 1 μm and the range of values less than or equal to 8μm.

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, reference to an “antibody” includes a single antibodyas well as two or more of the same or different antibodies, reference toan “excipient” includes a single excipient as well as two or more of thesame or different excipients, and the like.

II. System

In one aspect, a system comprised of a test device and an apparatuscapable of optically detecting a signal is provided. The test device andthe apparatus are designed for unique interaction with each other, aswill now be described. In the description below, the test device isexemplified by a lateral flow immunoassay, and is sometimes referred toas a test strip. It will be appreciated that the test device is notintended to be limited to the lateral flow immunoassay test device usedto exemplify the system, and a skilled artisan will appreciate thatother test devices, such as microfluidic devices, immunoassays otherthan lateral flow based immunoassays, are contemplated.

A. Apparatus

An embodiment of an apparatus capable of detecting a signal produced bya test device is illustrated in FIGS. 1A-1B. Apparatus 10 includes ahousing 12 that encloses an optics system, electronics software, andother components of the apparatus, all to be described herein below. Afront side 14 of the apparatus includes a user interface 16 that mayinclude, for example, a key pad 18 and a display screen 20. The key padincludes numeric keys for entry of numeric values, which can also belabeled with letters of the alphabet, a decimal point key, a back spacekey, and other keys that are desired by end users. As part of the keypad or as separate keys positioned elsewhere on the apparatus, thedevice may include keys to print test results, to advance printer paper,to open or close a drawer in the device, directional arrow keys and softor select keys for a user to interact and instruct the apparatus. In oneembodiment, the key pad is an alpha/numeric key pad, and the apparatusincludes a print key to activate print feature of a test result; a paperadvance key; navigation screen keys for a user to navigate through menuoptions displayed on interface screen (for example, right/left/up/downkeys, select keys).

The display screen can be, for example, a liquid crystal display screen,to receive output from a data processing unit in the apparatus anddisplay it to a user of the apparatus. In one embodiment, the displayscreen is a touch screen, for interaction with a user. An exemplaryscreen is a color screen with resolution of 320×240 (1/4 VGA) andadjustable contrast and brightness. Visible on the screen to a user willbe information such as test results, error messages, instructions,calibration information, troubleshooting information, user information,and the like.

An embodiment of the rear panel of apparatus 10 is shown in FIG. 1B andcan include port to receive a source of AC power 22 and an on/off toggleswitch 24, which in this embodiment is a soft key to activate thesoftware. The apparatus may additionally provide ports, on the rearpanel or elsewhere on the apparatus, to connect optional componentsand/or to interface with external instruments. For example, theapparatus may include a PS2 connector, for example, to interface with anexternal barcode reader; a port, such as an RJ45 port, to connect to alocal area network or Ethernet; a removable memory card port or slot;and/or a USB port. In a preferred embodiment, the apparatus includes aslot or port 26 for insertion of a removable non-volatile flash memorycard, such as an SD card, and the apparatus is capable of read and writeoperations to and from the SD card, to, for example, store all scan datafrom each test device, update system software.

With reference again to FIG. 1A, the apparatus can also include aprinter, such as a thermal printer, resident within the housing, and anopening 28 in a removable cover 30 on the housing is provided throughwhich paper from the internal printer exits the housing. The removablecover provides access to access or replenish a paper supply (not visiblein FIG. 1A) that interacts with the printer inside the apparatus.

The apparatus also includes a drawer 32 movable between open and closedpositions, as shown in FIG. 1A in its closed position and in FIGS. 2A-2Bin its open position. In the embodiment shown, the drawer is positionedon a front edge 34 of the apparatus, as seen best in FIG. 2B. It will beappreciated that the drawer could also be positioned on either side ofthe apparatus. In one embodiment, the drawer moves between its open andclosed positions by a mechanical mechanism, such as a latch and springmechanism. In one embodiment, the draw opens in response to a useractivating a key on the front or face of the apparatus, such as an “opendrawer” or “eject test device” button. In one embodiment, the drawer ismoved into its closed position after insertion of a test device manuallyby a user, or in response to a user activating a key or button on theapparatus. The drawer is configured to receive an immunoassay testdevice, further described below. Within the drawer, in one embodiment,is a distinct region, for example a depression, sized to receive thetest device. During operation of the apparatus, the test device remainsin a stationary position in the drawer, and therefore is positioned withprecision in the apparatus for precise interaction with a movable opticssystem, described below. Accordingly, the drawer comprises in oneembodiment a mechanism for positioning the test device for interactionwith the optics system. An exemplary embodiment of a positioningmechanism is illustrated in FIGS. 3A-3B.

FIG. 3A is an illustration of the internal components in the area of thedrawer in the apparatus, which are visible to a user only upon removalof the housing. In FIG. 3A, drawer 32 is in its open position, and atest device 36 is positioned in the drawer for insertion into theapparatus. A distal edge 38 of drawer 32 remains inserted within theapparatus when the drawer is in its open position, with a proximal edgeof the drawer being the portion of the drawer closest to a user and thatenters and exists the apparatus during use. Within the apparatus is areceptacle 40 into which drawer 32 can be received when the drawer ismoved into its closed position. Extending into receptacle 40 is apositioning arm 42 with a first end 44 and a second free end 46. Firstend 44 is movable within a track or slot 48 in the receptacle. The armis dimensioned and positioned such that its free end 46, or at least acorner of the free end, of the arm contacts an edge of the test device36 when the drawer is moving between its open and closed positions. Thisis apparent from the view shown in FIG. 3B, where the drawer is in itsclosed position and a corner of the free end of the arm is in contactwith the test device, and specifically with an edge of the test deviceexemplified by an optional housing surrounding a lateral flowimmunoassay. The arm via its contact with the test device gently pressesthe test device to a specific position within the drawer, and morespecifically within the slot in the drawer that is dimensioned toreceive and hold the test device.

Arm 42 is dimensioned and positioned to ensure precise lateralpositioning of the test device in the apparatus, more specifically,precise positioning in a plane that passes through the apparatus in aside to side direction with respect to the left/right sides of theapparatus. This plane is denoted by the x-x arrows in FIG. 1A. A secondarm can also be provided, to precisely position the test device along anaxis running from the front to back of the apparatus, referred to as alongitudinal axis and denoted by the z-z arrows in FIG. 1A. In oneembodiment, a second arm is located at a proximal end 50 of the drawer,to press against the test device to position it in the horizontal plane.The second arm, in one embodiment, is under tension by a spring, and inother embodiments is movable laterally and positioned to have a pressurepoint when in contact with a frontal edge of the test device.

Visible in FIGS. 3A-3B is a support rod 52 that extends at least thelength of receptacle 40. Support rod 52 provides a track along which amovable optics system in the apparatus travels, to scan the stationarytest device inserted into the apparatus. The optics system is nowdescribed with reference to FIG. 4.

A microprocessor-controlled optics system is positioned within thehousing of the apparatus such that it moves along the longitudinal axis(denoted by the z-z arrows in FIG. 1A) from a home or start position toa final position. The optics system includes an optics module comprisedof a carriage mounted on a track, the carriage movable by an electricmotor or actuator within the optics system of the apparatus. Secured tothe carriage, and part of the movable optics module, are an illuminationsource 54 and a detector 56, such as a photodiode. The illuminationsource can be mounted perpendicular to the test device and the detectoris oriented at an angle to collect emission from the test device. In theembodiment shown in FIG. 4, a photodiode is oriented at 40° relative tothe test device, and more generally the detector can be oriented at anangle of between about 20°-75° relative to the surface of the testdevice. In one embodiment, the optics module includes a single elementoptical detector (that is, an array of optical detectors is not present)and a single illumination source. The optics module can also compriseone or more filters, and the embodiment illustrated includes a filter58, and preferably a long pass filter, on the emission side of theillumination source, and a filter 60 positioned between the test deviceand the detector. In one embodiment, the illumination source emits UVlight at a wavelength that matches the excitation wavelength of a labelin the test device. In one embodiment, the illumination source is alight emitting diode (LED) that has a peak emission at 365 nm, moregenerally of between about 320-390 nm or 325-380 nm. In this embodiment,the long pass filter positioned in the optical path from the LED to thetest device transmits light between 310-315 nm.

In one embodiment, the photodetector is a broad band detector suitableto detect light at the wavelength emitted from the label in the testdevice. In one embodiment, the photodetector is a single-elementphotodetector (i.e., is not an array of photodetectors). In oneembodiment, the label is or contains a fluorescent, luminescent,chemiluminescent compound. As will be described below, an exemplaryfluorescent label is a lanthanide ion, such as europium, samarium,terbium and holmium, which each fluoresce at specific wavelengths.Filter 60 positioned in the optical path between the test device and thedetector, in one embodiment, transmits light above about 515 nm fordetection by the detector. A skilled artisan will appreciate that avariety of filters are known in the art (longpass, shortpass, bandpass,etc.) and can be selected according to the wavelengths of light desiredto excite a label and the wavelength of light desired for detection.

The optical system can also include an optical feedback loop. Theintensity or light output of the illumination source is controlled by afeedback loop using a monitor diode in the illumination path of theoptics. Power to the illumination source is adjusted based on lightoutput, where if, for example, light output decreases, current isincreased to compensate. In one embodiment, the power output from theillumination source is between 2-5 mW, more preferably between 2.5-5 mW.The feedback loop subsystem ensures consistent light output from theillumination source and reduces the frequency that calibration of theoptical system in the apparatus is required.

Resolution of the optics system to resolve or discriminate individual,discrete lines on the test device is a feature of the optics system, aswill become apparent from the description of the test device andoperation of the apparatus below. The illumination source preferablyprovides a focused beam of light capable of resolving one or more lineson a test device that are between 1-1.2 mm apart, or 4 mm apart measuredfrom the center of a first line and the center of an adjacent line. Inone embodiment, the shape of the beam of light from the illuminationsource is 2.5 mm by 0.8 mm, more generally is between 2-3 mm by 0.5-1.2mm. In another embodiment, the illumination source illuminates localizedregions of the detection zone on a test device (described below) and thesingle-element detector is synchronized with the illumination source inincremental movement along a movement path, thus permitting synchronizedillumination in a localized region and detection in the localizedregion. A field stop is provided to determine the shape of the beam oflight, and can be tailored according to the spacing between and width ofthe test lines on the test device. As will be described below, in oneembodiment, the spacing or tolerance between two adjacent test lines onthe test device and the shape of the beam of light are selected toprovide a dark space between test lines where no emissivity signaloccurs.

In one embodiment, the optical system comprises a splash shieldpositioned between the sample input on the test device and the opticalsystem, to protect the optics system and its movable module from liquidsample that may linger in the sample port/sample pad, particularly whenthe device is operated in walk-away mode, described below.

The apparatus, in some embodiments, includes a temperature sensingmeans, and in a preferred embodiment, includes at least two temperaturesensing devices housed within the housing of the apparatus. A firstinternal temperature sensor is positioned to detect the temperature inthe region associated with the optics system and a second internaltemperature sensor is positioned elsewhere in the apparatus away fromany internally generated heat source in order to detect ambienttemperature of the environment in which the apparatus is operated.

The apparatus includes internal memory storage with necessary softwarefor operation and for storage of data collected from sample analysis. Bythe SIM port or by an external computer (wireless or wired attachment),data can be exported from the apparatus or imported to the apparatus.

As mentioned above with reference to FIG. 1B, the apparatus is equippedwith ports for attachment to optional external devices, and an exampleis illustrated in FIG. 5. In this embodiment, the front or user side ofan apparatus 62 is shown, and attached to the apparatus is an externalbar code scanner 64. The bar code scanner interfaces with the apparatusvia a suitable data port provided on the apparatus. Externally attacheddevices ease transfer of data into and from the apparatus, and caneliminate user keyboard input, permitting accurate data input into theapparatus regarding a test to be analyzed or patient or sampleinformation. In one embodiment, a barcode scanner external is attachablevia PS-2 port on the apparatus and is capable of reading a linear or 1Dbar code.

In one embodiment, the apparatus is wireless or wired connected to adevice for delivering medical data to a third party such as the Centersfor Disease Control (CDC). In an exemplary embodiment, the apparatuscommunicates wirelessly with the 2Net™ Platform available from QualcommLife. The 2Net™ Platform is a cloud-based system that allows for thesecure transfer of data from the apparatus for storage. The 2Net™Platform gateways include, but are not limited to, a 2Net™ Hub, astand-alone FDA-listed external device, and a cellular componentembedded in the apparatus. Data from the apparatus is transferred to acloud via a 2Net™ Platform gateway where it can be stored, manipulated,and/or shared. In an exemplary embodiment, the data may be transferredto the CDC for reporting and/or surveillance of infectious agents. Inthis embodiment, it is preferable that the date be manipulated, such as“de-identified”, after transmission to the cloud storage to comply withapplicable rules and regulations such as HIPAA. It will be appreciatedthat the data may be transferred to one or more cloud storage sites(e.g. from a third-party cloud such as Qualcomm to a proprietary cloud).In one specific non-limiting embodiment, data from the apparatus iswirelessly transmitted to a Qualcomm cloud via the 2Net™ Hub. The datais then transferred to a proprietary cloud where it is “de-identified”to remove user information for compliance with HIPAA rules andregulations. The data is then transferred to the CDC for surveillance ofinfectious agents.

The apparatus can include additional optional features, including forexample acoustical output capability, to generate tones for audiblefeedback to a user, such as an error or test completion.

Calibration Cassette for Optics System

As described above, the apparatus comprises an optics system comprisedof an optics module that includes a light source, which in oneembodiment is an LED light source with a peak emission at 365 nm. Thedetector for the emitted fluorescent light from a label in a test deviceis a photodiode with filters to ensure that the light from thefluorescent reagent is not contaminated by ambient nor excitation light.Signal from the photodiode is translated through an analog to a digitalconverter where the digital signal is processed by a microprocessor inthe apparatus into a test result. To ensure consistent light output, theLED has a feedback loop whereby the optics system monitors the lightoutput of the LED and triggers an adjustment of the electrical currentto the LED to ensure a consistent intensity of the excitation light beamin real time. To further ensure that signal drift is controlled, theapparatus has a calibration algorithm that enables the user to insert acalibration cassette specifically designed for the apparatus andprovided with the apparatus. The calibration cassette is illustrated inFIG. 6.

FIG. 6 shows a calibration cassette 70 comprised of a calibration strip72 secured within an optional housing member, such as housing member 74which is separable in this embodiment into upper member 74 a and lowermember 74 b. A window 76 in upper housing member 74 a is provided sothat the optics system in the apparatus can interact with one or morelines on the calibration strip.

The calibration strip can comprise one or more lines, and in variousembodiments, comprises two or more lines, three or more lines or four ormore lines. In another embodiment, the calibration strip comprises atleast two lines, at least three lines, or at least four lines. Theembodiment illustrated in FIG. 6 shows a calibration strip with fourlines, identified as 78, 80, 82 and 84, and referred to herein below ascalibration lines or calibration test lines. The calibration lines arepositioned on the strip relative to the housing to be visible throughthe window when the strip is secured within the housing. In oneembodiment, the calibration strip is comprised of a material thatfluoresces upon excitation by light from the illumination source in theoptics system of the apparatus at a wavelength detectable by thephotodiode subsequent to passage through any filter(s) in the light pathof the photodiode. In one embodiment, the calibration strip is comprisedof a material that fluoresces, and the calibration lines are defined bymasking. For example, the fluorescing material can be silk-screened witha material that blocks light leaving the one or more calibration linesexposed. Alternatively, a fluorescing material can be deposited indiscrete lines onto a non-fluorescing material. In one embodiment, thefluorescing material in the calibration strip is a fluorescent whiteningagent deposited on or dispersed in a support material. Exemplaryfluorescent whitening agents optical brightener are dyes that absorblight in generally the ultraviolet and violet range (340-370 nm) of theelectromagnetic spectrum and re-emit light in the blue region (typically420-470 nm). Exemplary optical brighteners include compounds such asstilbenes (di-, tetra, or hexa-sulfonated), coumarins, imidazolines,diazoles, triazoles, benzoxazolines, biphenyl-stilbenes. A specificexemplary class of compounds are thiophenediyl benzoxazole compounds,and a specific exemplary fluorescent whitening agent is2,5-thiophenediylbis(5-tert-butyl-1,3-benzoxazole), an opticalbrightener. Exemplary support materials include polymers, andparticularly plastics, such as polymethylmethacrylates and polyesters,in particular biaxially oriented polyester. The whitening agent can bepolymerized with the support material during manufacture of thepolymeric support material, or can be deposited onto the polymericsupport after its manufacture. In a preferred embodiment, thefluorescing material forming the one or more calibration lines on thecalibration cassette fluoresces between 500-550 nm when excited.

The calibration cassette can optionally include a label, such as barcode 86 on the cassette in FIG. 6. In one embodiment, the bar code is atwo-dimensional bar code with information, for example, to confirm forthe apparatus that the cassette is a calibration cassette and withinformation regarding an expiration date for the cassette.

The calibration cassette is dimensioned to fit within the drawer of theapparatus, for interaction with the optics system, and in one embodimentthe apparatus and a dedicated, specific calibration cassette areprovided together as a kit. A user of the apparatus, typically whenprompted by the apparatus at a regular, defined period, such as every 30days, or once a month, or once every two months, etc., inserts thecalibration cassette into the drawer of the apparatus. The internal barcode reader within the apparatus transfers the information on thebarcode of the calibration cassette to the processor in the analyzer. Itwill be appreciated that the internal bar code reader is an optionalfeature, as the information on the bar code label can be entered intothe apparatus by a user using the key pad or via an external bar codescanner. From this information, the analyzer will confirm that acalibration cassette has been inserted into the analyzer, provide targetsignals the analyzer uses for comparison to actual signals obtained forthe calibration lines on the calibration strip, and provide theexpiration date of the calibration cassette. The analyzer then activatesthe optics system to initiate illumination of the calibration cassette,and specifically sequential illumination of each of the calibrationlines visible within the calibration cassette window. The analyzer thendetects the fluorescent signal from each of the calibration lines andstores the signal in memory. The detected signal for two of thecalibration lines is compared to the target (expected) signal for thatcalibration line. If the detected signal for each of two of thecalibration lines is within a predefined range of the target signal, forexample within (+/−) 1.75, 2%, 2.25%, 2.5% or 3%, then the calibrationof the analyzer is valid and no adjustments to the apparatus are needed.This calibration check event is recorded and stored in the memory of theapparatus.

If the detected signal for either or both of two of the calibrationlines is outside the predefined range of the target signal, but notoutside of a maximum predefined range, for example outside+/−3.25%,+/−3.5%, +1-3.75% or +/−4% of a predefined target signal for a specificline, the processor in the analyzer activates an algorithm toself-calibrate using a third or different calibration line on thecalibration test strip. Information for the target (expected) signalfrom this third line is also in the barcode information and was conveyedto the apparatus upon insertion of the calibration cassette and scanningof the bar code. When the signal for this third calibration line iswithin a defined acceptable range, the analyzer again reads the firsttwo calibration lines to confirm that the expected target signal isdetected for these two lines. If the signal is outside the maximumrange, the analyzer cannot recalibrate itself, and the system generatesan error message that is displayed to the user.

Apparatus Software

The apparatus includes an integrated software system used to collectdata from the lateral flow test assay, process the data, and display aresult to the user. The software can vary according to, for example, thedesign of the lateral flow test assay. An exemplary test assay isdescribed below along with software tailored to control the apparatus'interaction with the exemplary test assay. Here, a general descriptionof the software requirements is provided.

The software requirements are based on the test strip features andrequirements. These include the functional and non-functionalrequirements the software desirably meets in order to fulfill assayrequirements and user needs. The software specifications preferablyinclude three modes of user operation: Operator, Supervisor, andService. In addition to the main user modes, the software specificationspreferably include ethernet/Laboratory Information System (LIS)communication, printing, SD card interface and barcode scannerfunctionality. The software specifications preferably also provide poweron/off processing, battery, error handling, languages and audionotification. The software specifications preferably comprise thefollowing high level functions: analyzing fluorescent data from a testdevice, a calibration cassette, or a quality control test; selfcalibration; managing users for login; storing, managing, and recallingtest results; managing internal settings; printing results; sendingresults to a LIS; installing and operating in languages other thanEnglish; internal software checks, either at startup or continuously. Inone embodiment, the software specifications preferably check all or someof the following, either at startup or continuously: memory, powersupply; optics performance; stepper motor functionality; internaltemperature; internal clocks; and barcode reading. If errors occur, theyare logged in a message log and if applicable, the user is informed. Theapparatus is designed to recover from errors in a safe manner.

The apparatus software is based on 3-tier architecture, illustrated inFIG. 7. In brief, the software includes an application layer 90 thatfunctions to controls the system tasks. These are separate tasks thatrun in parallel and perform dedicated functions. This includes, forexample, controlling measurement scans, updating the graphical userinterface (GUI) screens, accepting keypad user input, printing andperforming remote communications. The tasks interact via message queues.A scheduler looks for tasks ready to run and activates them. Thesoftware also includes an abstraction layer 92 comprised of modulegroups that build up separate software subsystems. This builds a“convenience layer” for the application layer. The software sub-systemsinclude the data base subsystem, GUI objects, measured sequences, systemstatus and SDC transfer. The software also includes a hardware(HW)-driver level 94 comprised of modules for communication withhardware components of the system over an application programminginterface (API). The hardware components are the inter IC Bus (alsoknown as the I2C-Bus, this component facilitates communication betweenelectronic components), serial interface bus (SPI Bus), batteries,electronics, optional internal barcode reader and SD card.

As will be described further below, the software enables the apparatusto be operated in several modes, including a ‘read now’ mode where atest device inserted into the apparatus is immediately read; a ‘walkaway’ mode where a test device inserted into the apparatus is incubatedfor a selected or predetermined period of time prior to being read; amode to recall test results; a mode to recall control results.Accordingly, in one embodiment, the apparatus is designed to be operatedin two or more, three or more or four or more modes.

Test Device: Exemplary Lateral Flow Immunassay

With reference to FIG. 8 and FIGS. 9A-9B, an embodiment of a test devicefor interaction with the apparatus is illustrated. The test device isexemplified in the drawings below by a lateral flow test immunoassay,however it will be appreciated that a lateral flow immunoassay isexemplary of test devices suitable for interaction with the apparatus.Test device 100 is comprised of, in sequence, a sample pad 102, a labelpad 104, one or more lines indicated collectively at 106 and selectedfrom a test line, a control line and a reference line, and an absorbentpad 108. In one embodiment, a support member 110 is provided, and eachor some of the sample pad, label pad, lines and absorbent pad aredisposed on the support member. As will be described below withreference to FIG. 10, the test device comprises a region between thedownstream edge of the most downstream analyte-specific test line, whichin the embodiment shown in FIG. 8 is test line for binding to aninfluenza antigen (e.g., a test line that comprises anti-flu Aantibodies), and the upstream edge of the absorbent pad 108 is aprocedural control zone, denoted PCZ in FIG. 8. In some embodiments, thetest device additionally includes a desiccant portion, not shown in FIG.8, but visible in an embodiment shown FIG. 9A. A desiccant portion canbe positioned on the support member of the test device, and in oneembodiment is disposed on the support member downstream of the absorbentpad, as described in U.S. Patent Application Publication No.2008/0311002, incorporated by reference herein. In another embodiment,seen in FIG. 9A, a desiccant portion 112 is a discrete component,physically separate from the test strip, inserted into a housing memberthat contains the test strip.

In one embodiment, the test strip is enclosed in a housing, sometimesreferred to as a cassette, such as housing 114 in FIG. 9B. Optionalhousing 114 in this embodiment is comprised of an upper member 116 and alower member 118 that fit together to form a housing. Lower member 118may include architectural features that define dimensioned regions forreceiving the test strip 110 and the optional desiccant 112. Upperhousing member 116 includes at least two openings, a first sample inputport 120 and a viewing window 122. The sample input port is disposeddirectly above the sample pad on the test strip, so that a sampledispensed into the sample input port contact the sample pad for flowalong the test strip. In the embodiment shown, the sample input portincludes a bowl portion to receive a liquid sample into the port. Theviewing window is positioned to reveal the lines in the test strip, sothe optics system in the apparatus can interact with the lines, as willbe described below.

In the embodiment shown in FIG. 9B, a bar code label 124 is affixed tothe upper housing member. It will be appreciated that the bar code labelcan be positioned elsewhere on the housing, and is positioned forinteraction with the internal bar code scanner positioned within theapparatus. In one embodiment, the bar code label is a 2D bar code,encoding information, for example, regarding the assay test strip, suchas the pathogen/analyte the test strip is designed to detect (Flu A/B,Strep A, RSV, others listed below, etc.) which informs the apparatuswhat protocol in memory to initiate for scanning the test strip; aunique test serial number so that the apparatus will not read same teststrip twice. In one embodiment, the information contained in the barcode does not include information related to the patient or the sampletype, and is limited to information about the test strip.

It will be appreciated that the test device illustrated in FIGS. 8-9 isexemplary of lateral flow test devices in general. The test strip can beconfigured uniquely for any given analyte, and the external housing isoptional, and if present, need not be a cassette housing but can be aflexible laminate, such as that disclosed in U.S. Patent ApplicationPublication No. 2009/02263854 and shown in Design Patent No. D606664,which are both incorporated by reference herein. The system requiresonly that the drawer in the apparatus and the test device be dimensionedto receive the test device in the drawer, and the optics system in theapparatus have a movement path the scans the necessary regions of thetest device.

In that regard, FIG. 10 is a top view of an exemplary test strip and thearrangement of its structural and immunochemical features forinteraction with the apparatus. Test strip 130 includes a samplereceiving zone 132 in fluid communication with a label zone 134. A fluidsample placed on or in the sample zone flows by capillary action fromthe sample zone in a downstream direction, indicated by arrow 135. Labelzone 134 is in fluid communication with at least a test line and acontrol line or a reference line. In the embodiment shown in FIG. 10,the label zone is in fluid communication with a negative control line136, an analyte test line 138, an optional second analyte test line 140,a reference line 142. The two or more lines are in fluid communicationwith an absorbent zone 144. That is, the label zone is downstream fromthe sample zone, and the series of control and test lines are downstreamfrom the label zone, and the absorbent pad is downstream from theportion of the test strip on which the lines are positioned. A regionbetween the downstream edge of the most downstream analyte-specific testline, which in the embodiment shown in FIG. 10 is test line 140, and theupstream edge of the absorbent pad is a procedural control zone (PCZ)146. Reference line 142 is within the procedural control zone 146. Aswill be described below, the procedural control zone, and in particularthe reference line therein, (i) ascertains whether sample flow along thetest strip occurred based on its RLU signal (emission), and (ii) is usedby the algorithm to determine the relative locations of the other lines(control, if present, and analyte-specific test line(s)) on the teststrip. Materials for construction of each of the zones is well known inthe art, and includes, for example, a glass fiber material for thesample zone, a nitrocellulose material on which the two or more linesare positioned.

The sample zone receives the sample suspected of containing an analyteof interest control its flow into the label zone. The label zone, in oneembodiment, contains two dried conjugates that are comprised ofparticles containing a lanthanide element. The lanthanide materialsinclude the fifteen elements lanthanum, cerium, praseodymium, neodymium,promethium, samarium, europium, gadolinium, terbium, dysprosium,holmium, erbium, ytterbium, lutetium, and yttrium. In one embodiment,the particles are polystyrene particles or microparticles (particlesless than about 1,000 micrometers in diameter, preferably less thanabout 500 micrometers in diameter, more preferably less than 200, 150 or100 micrometers in diameter) containing a luminescent or fluorescentlanthanide, wherein in one embodiment, the lanthanide is europium. In apreferred embodiment, the lanthanide is a chelated europium. Themicroparticles, in one embodiment, have a core of a lanthanide materialwith a polymeric coating, such as a europium core with polystyrenecoating. A binding partner for the analyte(s) of interest in the sampleis/are attached to or associated with the outer surface of themicroparticles. In one embodiment, the binding partner for theanalyte(s) of interest is an antibody, a monoclonal antibody or apolyclonal antibody. A skilled artisan will appreciate that otherbinding partners can be selected, and can include complexes such as abiotin and strepavidin complex. Upon entering the label zone, the liquidsample hydrates, suspends and mobilizes the dried microparticle-antibodyconjugates and carries the conjugates together with the sampledownstream on the test strip to the control or reference and test linesdisposed on the nitrocellulose strip. If an analyte of interest ispresent in the sample, it will bind to its respective conjugate as thespecimen and microparticles flow from the label zone onto the surface ofthe nitrocellulose. In the embodiment shown in FIG. 10, this flowingmixture will then encounter negative control line 136. The negativecontrol line is comprised of mouse immunoglobulin (IgG) and enablesdetection of non-specific binding of the conjugates to theimmunoglobulin, thus approximating the level of non-specific bindingthat will occur at the downstream test line(s). The signal generated atthis negative control line is used to help ensure that high non-specificbinding at the analyte-specific test line does not lead to falsepositive results.

As the sample and microparticle-antibody conjugates continue to flowdownstream, if antigen is present in the sample, the fluorescentmicroparticle-antibody conjugate which now includes bound withantigen/analyte of interest, will bind to the test line(s). In someembodiments, a single test line is present on the test strip. In otherembodiments, at least two, or two or more test lines are present on thestrip. By way of example, and as detailed in Example 1, a test stripintended for detection and/or discrimination of influenza A andinfluenza B will include a first test line to detect influenza A and asecond test line to detect influenza B. Microparticle-antibodyconjugates comprised of microparticles coated with antibodies specificfor influenza A and microparticles coated with antibodies specific forinfluenza B are included in the label zone. A first test line forinfluenza A and a second test line for influenza B are disposeddownstream of the label zone, and preferably downstream of the negativecontrol line. The first test line for influenza A comprises a monoclonalor polyclonal antibody to a determinant on the nucleoprotein ofinfluenza A and the second test line for influenza B comprises amonoclonal or polyclonal antibody to a determinant on the nucleoproteinof influenza B. If antigen is present in the sample, a typicalimmunoassay sandwich will form on the respective test line that matchesthe antigen in the sample.

The microparticle-antibody conjugates that do not bind to the negativecontrol line or to a test line continue to flow by capillary actiondownstream, and the remaining sample encounters the reference line. Thereference line is comprised of goat anti-mouse immunoglobulin, and atleast a portion of microparticle-antibody conjugates that reach thereference line will bind non-specifically to the goat anti-mouseimmunoglobulin. Fluorescent signal generated at this line providesinformation, for example, about the flow of the sample and also canserve as a location marker to direct the apparatus to the precise otherlocations on the nitrocellulose that are to be scanned by the opticssystem, as will be described below. The remaining sample then flowsdownstream of the reference line into the procedure control zone 146that is also scanned by the optics system and is used, for example, toconfirm that adequate flow of the sample has occurred. The sample withany remaining microparticle-antibody conjugate then flows on into theabsorbent pad.

III. Test Procedure and System Operation

As mentioned above, the apparatus can be operated in two modes, a‘read-now’ mode or a ‘walk-away’ mode. The flow of sample fromdeposition on the sample pad to the absorbent pad takes between 2-20minutes, more typically between 5-18 minutes, more typically between7-15 minutes. A user can opt to place the sample on the sample pad ofthe test device, insert the test device into the drawer of theapparatus, and set the apparatus in ‘walk-away mode’ whereupon theapparatus will scan the bar code on the test device and determine thecorrect incubation time for the test device that permits sufficient timefor the sample to flow from the sample pad to the absorbent pad.Alternatively, a user can opt to place the sample on the sample pad ofthe test device and incubate the test device external the apparatus. Thetest device is then inserted into the drawer of the apparatus subsequentto an incubation period external to the apparatus, and a user canoperate the apparatus in ‘read-now’ mode wherein the apparatus will scanthe bar code on the test device to determine the assay type andimmediately initiate the scanning protocol for that assay type. Thescanning protocol and the processing of information from a scan will nowbe described, with reference to FIGS. 11-12.

A. Operation of Apparatus

To initiate a scan of a test device, the apparatus is powered-on ifneeded and the toggle switch to initiate the apparatus software isactivated. Prior to inserting the test device with sample into theapparatus, using the optional external bar code reader information aboutthe user, the sample, the patient, etc. can be scanned into theapparatus memory. With reference to FIG. 11, a “start test” button onthe apparatus or on the touch screen is pressed, 150, to start ameasurement of a test device. The apparatus takes a temperature reading,152, and then automatically opens the drawer in the apparatus, 154, toreceive the test device on which a sample has been dispensed onto thesample pad. The test device with loaded sample is inserted into thedrawer, 156, and the drawer is closed manually, 158, with gentlepressure by the user. As the drawer closes, one or more positioning armspress against the test device to position it in the drawer in a preciselocation that is consistent from test to test. The optics shield withinthe apparatus is positioned to protect the optics system and its movableoptics module from any liquid sample that may splash from the sampleinput port when the drawer closes.

Closure of the drawer initiates a sequence of events, 158, comprised ofthe following. The internal bar code reader scans the bar code on thetest device and receives information regarding the assay type (e.g.,influenza A/B, Strep A, RSV, etc.), the serial number and the expirationdate of the test device, optical cut-off information for the assay type,and any other information included on the bar code secured to the testdevice. In one embodiment, a mirror is positioned to facilitateinteraction of the light beam from the internal bar code scanner and thebar code label on the test device. It will be appreciated that theinternal bar code reader is an optional feature, as the information onthe bar code label can be entered into the apparatus by a user using thekey pad or via an external bar code scanner. Based on the test assaytype discerned from the information on the bar code label or otherwiseprovided to the apparatus processor, the apparatus initiates analgorithm stored in the apparatus' memory for the assay for which thetest device is designed, and based on user defined selection of read-nowmode or walk-away mode, a protocol stored in memory initiates. Inwalk-away mode the apparatus incubates for a period of time, 160, priorto initiating a scan of the test device, 162; in read-now mode theapparatus does not wait for the preset incubation time for thatparticular assay, and immediately begins a scan of the test device, 162.

The scan and evaluation of the test device, 162, comprises anothertemperature check, 164, at the same or different position from the firsttemperature check 152. The initiated algorithm activates the opticssystem, including the stepper motor that moves the optics module withrespect to the test device that is stationary in the apparatus. Theoptics system searches for its home position, 166, (described below) andthen conducts a scan of the measurement window, 168, in the housing ofthe test device through which the reference/control and test lines arevisible. The motor in the optics system moves the optics moduleincrementally from a defined start point along the length of themeasurement window in the test device in accord with parameters definedby the algorithm for the particular assay being conducted. As will bedescribed in more detail below, the optics module is moved inincremental steps by the motor in the optics system along the length ofthe test window, in a downstream to upstream direction with respect tosample fluid flow on the test strip, wherein the optics carriage stopsat each incremental step or position to illuminate the position, detectemitted light after illumination at that position, before advancingupstream to the next position.

After collection of emitted light at each of the plurality ofincremental positions along the length of the test window, the algorithmlocates and evaluates the data in the data array that is associated withthe control lines, 170, and conduct a qualitative analyte evaluationusing a cut-off algorithm, 172.

The algorithm then determines whether the test is a clinical test, anexternal control or a calibration test, 174, and if the determination isyes (based on information provided on the test device bar code or basedon user input information), the results are stored to memory, 176, suchas on the SD card or in the apparatus memory. An intermediate scan 178may be performed after the results are stored on the SD card. If thetest is not a clinical test, then results are stored to flash memory,180, and displayed and/or printed, 182. The drawer is then opened, 184,by the apparatus or by the user at the end of the measurement sequencefor the user to remove the test device, 186.

FIGS. 12A-12B shows a second exemplary test sequence for the apparatusdescribed herein. It will be appreciated that the test sequence iseasily varied by simply varying the programming in the software programsin the device, to alter the sequence of events, time allocated to eachevent, etc., in a measurement procedure. In the exemplary procedure ofFIGS. 12A-12B, start of the measurement procedure is initiated, 190, bypressing a button or switch on the apparatus. Information regarding thepatient (name, gender, age, etc.) is entered, 192, by the user via entryusing the keypad on the apparatus or via an external bar code scanner.The drawer in the apparatus is opened using a button on the apparatusand the test device is inserted into the drawer, 194. Closing of thedrawer manually or automatically by the apparatus initiates an automatedsequence of events, 196. The sequence includes reading the temperatureat one or more locations inside the apparatus, such as adjacent the testwindow, and/or taking an ambient temperature reading, 198. If anincubation time is commanded by a user selecting ‘walk-away’ mode or bya pre-programmed requirement for a particular test assay, an incubationtime starts, 200. Upon completion of the incubation time or if noincubation time is required or commanded, the measurement sequence bythe optics system automatically initiates, 202.

With reference now to FIG. 12B, the measurement sequence by the opticssystem includes activating the motor that moves the optics module, 204,and the optics module finding its home position, 206. A temperaturereading, 208, in the vicinity of the optics system can be taken. At afirst position along the optical read path that corresponds with thetest window on the test device inserted in the apparatus, theillumination source in the optics module is turned on and then off, andduring the off period fluorescent emission is detected by thephotodetector in the optics module. The detected emission is stored inmemory, and the motor in the optics system advances the optics module afixed amount to its next position, which in an embodiment is in adirection toward the sample zone in the device so that measurement ofthe lines in the test window occurs in a downstream to upstreamdirection with respect to fluid flow on the test strip. After completionof a predefined number of incremental steps along the length of the testwindow and capture of light emission at each step, 210, the opticsmodule is returned to its home position by the motor, 212, and the motoris powered off, 214. It will be appreciated from this description that,in one embodiment, the apparatus comprises a dynamic optics module of anillumination source and a photodetector, wherein the module is staticduring an illumination/detection sequence and resumes dynamic movementthereafter. It will also be appreciated that the dark reading, i.e,detected emission during the off, or dark period, of theillumination-detection sequence, is utilized for purposes of baselineand background and not for time-resolved fluorescence.

The algorithm stored in apparatus memory for that particular assay thensearches the data array for the peak emissions for each of the test,control, reference lines, 216, to calculate line intensities of peakarea or peak height, 218. The algorithm calculates results from the dataarray, 220, and stores the results to memory, such as on the SD cardinserted into the device. The calculated result can be displayed to thescreen on the apparatus, or prompted to be printed by the user, orstored in flash memory if needed, 222. A user can then instruct theapparatus to open the drawer, to remove the test device, 224, 226,ending the measurement procedure, 228.

In one embodiment, and with specific reference to a test device likethat shown in FIG. 10 for detection and/or discrimination of twoanalytes in sample, such as influenza A and influenza B, the strip isincubated in the apparatus when operated in walk-away mode forapproximately fifteen minutes, at which time the apparatus initiates anoptical scan of the test device, measuring fluorescence signals acrossthe strip's length and performs calculations and reports the testresults. The scanning of the test device strip and analysis requiresless than about 60 seconds, and more preferably is between about 20-60seconds, more preferably 30-45 seconds.

The apparatus, the test device, and the test procedure wherein the twointeract, has several built-in control features to ensure that thecorrect result will be reported apparatus. A first feature is thelocation of the reference line on the test device, which is used by thealgorithm to determine the relative locations of the other control andanalyte-specific test lines on the test device. The software programexpects the reference line to be located within a specific, pre-definedlocation range. The acceptable range for the location of the referenceline is based on the manufacturing tolerance for placement ofimmunochemistry on the test strip, for location of the test strip withinthe housing, and for positioning of the test device (test strip in thehousing (cassette)) within the drawer of the apparatus. Once thereference line is located, the positions of each of the other test linesand zones are determined by the algorithm and match the locations of thevarious chemistries deposited on the test strip.

More specifically, and with reference to FIGS. 13A-13C, a top view of atest device 230 is shown, the test device having an external housing232. Inserted inside the housing is a test strip 234, seen partially inFIG. 13A through the window 236 in the housing. The portion of teststrip 234 comprising the at least one control or reference line and atleast one test line, also referred to herein as the “nitrocelluloseregion” of the test strip, is shown in FIG. 13C. A reference line 238 onthe test strip is the distal most line on the test strip, relative tothe proximal end of the test strip where the sample pad is positioned.That is, the reference line is the line furthest downstream (withrespect to the direction of sample fluid flow) on the test strip.Because the optics system scans the nitrocellulose region visiblethrough the test window in the housing in a downstream to upstreamdirection, as indicated by arrow 240 in FIG. 13C, where downstream toupstream is relative to sample fluid flow on the test strip, thereference line is the first line encountered with the optics systeminitiates its scan of the test device. As mentioned above, the referenceline is comprised of, for example, goat anti-mouse polyclonal antibody,or another antibody that provides non-specific binding. The referenceline serves two purposes. First, the reference line's relativefluorescence units (RFU) signal desirably exceeds a specified minimum(such as 1,000 RFUs) in order to demonstrate adequate sample flow,otherwise the test is interpreted as invalid. The minimum RLU isinformation that can be provided on the bar code for each test device oris information stored in memory. It will be appreciated that the minimumRLU for a reference line in a test strip may be assay specific, wherethe minimum RLU for a test device for Strep A may be different than theminimum RLU for a test device that detects RSV. Second, the location ofthe reference line peak in the data array enables the use of analgorithm to locate the other test lines on the test strip, andspecifically in the nitrocellulose region, thereby specifying thescanning locations for the other test lines.

The test strip also includes, in some embodiments, a negative controlline 242. The negative control line is comprised of normal mouseimmunoglobulin (IgG), and provides two functions. The negative controlline enables measurement of the level of non-specific binding of themicroparticle-antibody conjugates, thereby approximating the level ofnonspecific binding that will occur on the test line(s). The negativecontrol line also serves as an indicator of adequate incubation time andsample flow; e.g. if the RLU signal for the negative control lineexceeds a specified maximum, adequate flow has not yet occurred and theassay is interpreted as invalid.

The test strip also includes at least one analyte-specific test line. Inembodiments where two or more analytes are to be detected anddiscriminated, two or more test lines are provided. In one embodiment,such as the embodiment illustrated in FIG. 13A and FIG. 13C, the teststrip comprises two analyte-specific test lines, for example, a firstline 244 with monoclonal antibodies to the nucleoprotein of influenza Aand a second line 246 with monoclonal antibodies to the nucleoprotein ofinfluenza B. These analyte-specific test lines serve to capture thefluorescent microparticle-antibody conjugates when in the presence oftheir corresponding, respective antigens.

The tolerance between lines (analyte-specific, reference and control) onthe test strip is precise, so that the optics module illuminatesdirectly above a middle region of the lines. The reference line in someembodiments is wider that the other lines on the test strip, to give theoptics module a larger target for finding its point of reference. Itwill be appreciated that the lines require a certain minimum spacing toavoid overlap of peak emission so that a baseline can be determinedbetween each line. As seen in FIG. 13B, which shows a cross-section ofthe window of a test device, the angle of the wall 248 of the testwindow 236 creates a shadow 250. The shadow decreases the actual windowlength to an effective window length. The reference line 238 has acertain width w_(ref), that in one embodiment is larger than the widthw_(test), of an analyte-specific test line. The reference line has anupstream edge 252 and a downstream edge 254, with respect to thedirection of fluid flow on the test strip. A procedural control zone 255is defined by the downstream edge 254 of the reference line and thedownstream end 256 of the effective window length. In one embodiment,the dimensional width of the procedural control zone 255 is less thanthe width between lines upstream of the reference line. For example, inthe embodiment shown in FIG. 13C, test line 244 has a downstream edge258 and test line 246 has an upstream edge 260. The distance betweenedges 258 and 260 defines a width, w_(l1→l2), between lines upstream ofthe reference line, and in one embodiment this width, w_(l1→l2), isgreater than the width of the procedural control zone. The spacingw_(l1→l2), between two lines in the nitrocellulose region of the teststrip, is determined in conjunction with the shape of the beam of lightfrom the LED such that a dark space between test lines where noemissivity signal occurs. This ensures a baseline is detected betweeneach peak emission from the sequential, adjacent test lines.

The optics system in the apparatus is an assembly of mechanical,electronic and optical components which serves to illuminate the teststrip with an ultraviolet light-emitting diode (UV LED) and thencollects, processes and transforms the resulting europium fluorescencesignal using a photodiode to an electronic signal that is converted byan analog-to-digital converter into useable analytical data. The LED isa semiconductor device which emits light (UV at 365 nm in oneembodiment), and during a measurement procedure the LED is pulsed on andoff at each incremental step of the optics system along the optical pathdefined by the test window. The resulting fluorescence emission iscollected by a photodiode during and between illumination pulses. Theunprocessed signal is manipulated by subtracting the background signal(LED off) from the signal with the LED on. The optic system collectssignal data expressed in RFUs from each of the lines (reference, test,and control), when the optics module is positioned at one of isplurality of incremental positions along the length of thenitrocellulose region on which the plurality of lines are disposed. Inone embodiment, the dwell time of the optics module at each incrementalstep is on the order of 2000-8000 microseconds, more generally between3000-4500 microseconds. It will be appreciated that the dwell time ateach step can be varied to manipulate sensitivity of the assay, where alonger dwell time at each step will increase sensitivity, and dwell timecan be decreased to decrease overall test time.

At end of a scan of lines in the window, the data collected consists ofemission signals from the test strip at the position when LED is on (365nm) and emission signals from test strip at that position when LED isoff. The difference between these values at each position is taken, andstored in memory as a one-dimensional array of the difference inemissivity at each position when LED is on and when LED is off. The dataprocessing algorithm smooths the data array using a local polynomialregression (of degree k) on the series of differential emissivity valuesequally spaced in the series (such as the Savitzky-Golay method), todetermine a smoothed value for each point. In one embodiment, thealgorithm smooths 13 points in the array, and the first derivative ofthe smoothed data array is taken, and the peak/trough of the derivativepeak height is determined to be the cut-off value. In other embodiments,the apparatus is programmed to analyze the resulting signal data in away that will yield qualitative results. The programming involves acut-off value that corresponds to an analyte-specific RFU signal that isfirst defined by the apparatus' algorithm and then used to helpcalculate a positive or negative result. The signal to cut-off ratiocorresponds to a simple ratio of the signal in RFUs obtained at eachtest line divided by its cutoff where the specimen giving any S/CO value≥1 is positive and any S/CO value <1 is negative. Signal data that iscollected is processed using a Savitzky-Golay smoothing algorithm thatuses a weighted average smoothing method that reduces unwantedelectronic noise, while preserving actual test signals, includingmaxima, minima and peak width with little impact on their actualdimensions.

In one embodiment, a negative control threshold is provided on the barcode information for each test device. The negative control threshold isa test strip lot-specific RFU value that impacts the calculation of thecut-off. For example, when the RFU of the negative control exceeds thenegative control threshold value, then the cutoff calculation is changedfrom a fixed cutoff of 675 RFUs to a cutoff based on multiplying the RFUof the negative control value found on each test strip by a lot-specificcorrection factor. In another embodiment, a dual positive thresholdvalue is provided for each test device, which corresponds to an RFUlevel used to determine the clinical outcome for a sample when the RFUsignals for antigen A (e.g., influenza A) and for antigen B (e.g.,influenza B) are both above their respective cutoffs—i.e. when a dualpositive result is obtained. After the cutoffs for antigens A and B aredetermined, the RFU value of the antigens A and B test line signals arecompared to this dual positive threshold value. Depending upon thiscomparison, a special algorithm may be triggered to calculate a positiveor negative test result.

FIGS. 14A-14C are graphs showing an exemplary data set from an opticalscan of a test device for detection of influenza A and influenza B. Uponcompletion of a scan of the test strip, a signal differential isdetermined for each incremental position by taking the differencebetween the signal detected during the illumination measurement wherethe LED is on (s(i)_(illum)) and the signal detected during darkmeasurement where the LED is off (s(i)dark). This ‘dark-correctedsignal’, s(i)DC, at each position i is calculated by:sDC=sillum−sdarkThe dark-corrected signal is checked for consistency by testing thecondition:sDC(i)>MinDarkCorrCounts for all iwhere MinDarkCorrCounts corresponds to the minimal allowed value for thedark corrected signal. Conversion of signal index (i) to a position x inmm is done by:x _((i)) =x _(start) +Δx·ii _((x))=roundtonearest(x−x _(start) /Δxwhere x_(start) is the start position of the scan, x is the distancebetween two samples of the signal in mm.

Signal preparation is done by smoothing the signal and calculating thefirst derivative. Both the smoothed signal and the derivative of thesmoothed signal are used as input parameters for a peak analysis. Thefirst line in the scan, which corresponds to the reference line, is theposition reference line for the other lines on the test strip. Thereference line has a wider search range than the other lines, giving ita larger tolerance. Based on this positional control, the expectedposition (x) of the other peaks in the data set is known. The algorithmconducts a polarity check on the derivative of the peaks correspondingto the analyte-specific test line and control lines, to determine if thepeak is a maximum and not a minimum. As seen in FIG. 14B, the derivativeof a positive peak (maximum) has a maximum, a zero crossing, and aminimum. The polarity check considers two points of the derivative; oneis located half peak width (expected) left of the (expected) peakcenter; the other is half peak width right to center. If these pointsare connected by a straight line, this line must have a negative slope.A polarity check of the reference line is not done, as the positionaltolerance for the reference control line is high. The next step in peakdetection is searching for a maximum and a minimum in the derivative,which is illustrated in FIG. 14B. Then, a peak height can be calculated,as shown in FIG. 14C. The algorithm calculates the peak height against abaseline, and if the peak height is greater than a cutoff value, theanalyte is present and a positive result is reported.

In summary, the apparatus and test device described herein includeseveral features that are uniquely interactive to provide a sensitive,specific system. The test device includes a label pad with particles ormicro-beads having a fluorescent material, and coated with antibodieswith specific binding affinity for an analyte of interest. The testdevice includes a procedural control located after the lastanalyte-specific test line on the test strip. The procedural controlzone is a region located between the last analyte test line and theabsorbent pad. The analyzer scans this zone positioned at the downstreamend of the test device, and determines whether adequate flow of thesample has occurred. A minimum and maximum fluorescent signalspecification for this procedural control zone is one of the fail safefeatures incorporated into the assay. No colored test or proceduralcontrol lines will be visible to the human eye in the test window of thefluorescent assay cassette. The apparatus automatically scans the teststrip, collects and analyzes the fluorescence data, and then calculatesand reports the result. These features eliminate the subjectivityrequired to interpret results in visually human-read lateral flowassays. In addition, a negative control line located on the test strip,downstream of the label pad, and before an analyte specific test line.This negative control line serves as another procedural control and alsoas a source of information for calculating each assay's cutoff. Theassay's sensitivity is derived from the use of a unique polystyrenemicrobead that has been dyed with a chelate of europium. The europiumcompound (more than 1×10⁶ fluorescent molecules per bead) that isencased within the microbeads is temperature stable, resistant tobleaching in room light, and yields a very efficient conversion of theUV energy from 365 nm to a wavelength of 618 nm. This large Stokes shiftprotects against many naturally occurring fluorescent compounds that maybe present in the test materials and/or clinical specimens.

B. Assays and Analytes to be Detected

The system comprised of an apparatus and a test device as describedherein is intended for detection of any analyte of interest. Analytesassociated with a disorder or a contamination are contemplated,including biological and environmental analytes. Analytes include, butare not limited to, proteins, haptens, immunoglobulins, enzymes,hormones, polynucleotides, steroids, lipoproteins, drugs, bacterialantigens, viral antigens. With regard to bacterial and viral antigens,more generally referred to in the art as infections antigens, analytesof interest include Streptococcus, Influenza A, Influenza B, respiratorysyncytial virus (RSV), hepatitis A, B and/or C, pneumoccal, humanmetapneumovirus, and other infectious agents well known to those in theart.

In other embodiments, a test device intended for detection of one ormore of antigen associated with Lyme disease,

In another embodiment, a test device designed for interaction with theapparatus is intended for use in the field of women's health. Forexample, test devices for detection of one or more of fetalfibronectin,chlamydia, human chorionic gonadotropin (hCG), hyperglycosylatedchorionic gonadotropin, human papillomavirus (HPV), and the like, arecontemplated.

The test devices are intended for receiving a wide variety of samples,including biological samples from human bodily fluids, including but notlimited to nasal secretions, nasopharyngeal secretions, saliva, mucous,urine, vaginal secretions, fecal samples, blood, etc.

The test devices, in one embodiment, are provided with a positivecontrol swab or sample. In another embodiment, a negative control swabor sample is provided. For assays requiring a external positive and/ornegative control, the apparatus is programmed to request a user toinsert into the apparatus a test device to which a positive controlsample or a negative control sample has been deposited. Kits providedwith the test device can also include any reagents, tubes, pipettes,swabs for use in conjunction with the test device.

IV. Examples

The following examples are illustrative in nature and are in no wayintended to be limiting.

Example 1 Detection and Discrimination of Influenza A and B

A lateral flow test device comprised of a test strip and a housing wasprepared. The test strip was fabricated to have a sample pad comprisedof a glass fiber matrix in fluid connection with a nitrocellulose strip,one or both supported on a support membrane.

Using standard NHS/carboxyl chemistry, specific monoclonal antibodieswere covalently bound to the surface of europium chelate(β-diketone)-incorporated polystyrene beads to form fluorescentmicroparticle-antibody conjugates. The microparticle-antibody conjugateswere deposited on a glass fiber matrix to form a label pad. The labelpad was positioned adjacent the sample pad in a downstream direction.Two populations of microparticle-antibody conjugates coated withuniquely different monoclonal antibodies, one with monoclonal antibodydirected to influenza A nucleoprotein and a second with monoclonalantibody directed to influenza B nucleoprotein, were prepared anddeposited in the label pad.

An absorbent pad comprised of a highly absorptive material that acts asa wick to draw fluid from the nitrocellulose strip, thereby helping toensure that adequate sample flow through the entire test strip wasachieved, was positioned on the test strip downstream from the label padand the nitrocellulose region.

The test strip was secured in a housing, for ease of handling. On anexternal upper surface of the housing was a bar code label containinginformation about the test strip, including for example, the intendedanalyte to be detected (influenza A and influenza B), a device specificidentification number, and an expiration date.

A nasal swab sample from a patient presenting with flu symptoms wastreated with a reagent solution to form a test mixture. A portion of thetest mixture was dispensed onto the sample pad via the sample input portin the housing.

Using the external barcode reader, the user scanned the patientinformation into the apparatus or enters the information using thekeypad on the apparatus. The user selected walk-away mode and insertedthe test device into the drawer of the apparatus. After an incubationtime of 15 minutes, the apparatus initiated its measurement sequence toscan the test device. The internal bar code scanner read the informationon the bar code label on the test device to determine the assay type,the device lot number, the test device serial number and the test deviceexpiration date. The microprocessor loaded the correct program intomemory for the assay type to be run.

The microprocessor-controlled optics unit in the apparatus conducted itsincremental, step by step scan of the length of the viewing window,which approximately corresponds to the length of the nitrocelluloseregion on the test strip. On the nitrocellulose strip the lines weresequentially read, beginning with the most downstream line, thereference line. The optics module moved relative to the stationary testdevice in an upstream direction to each of the analyte-specific testlines. At each incremental step, UV light from the UV LED with a peakemission at 365 nm was flashed on and then off. The UV light excited theeuropium fluorophore which in turn emitted light at a wavelength of 618nm.

After the apparatus completed its optical scan of the test window on thetest device and collected the fluorescent data, it objectivelyinterpreted the assay result. There were five possible results: (1)positive for influenza A and negative for B; (2) positive for influenzaB and negative for A; (3) positive for both influenza A and B; (4)negative for both influenza A and B; and (5) invalid. A positive resultfor either analyte was determined by detection of a fluorescent signalat levels above a signal threshold set upon scanning the negativecontrol line by a specific algorithm in the apparatus. The fluorescencesignal obtained with this assay was invisible to the unaided eye, andindicated the test sample was positive for influenza A and negative forinfluenza B. The test result can only be obtained with a fluorescentanalyzer, which afforded fully objective interpretation of the testresult.

Example 2 Detection and Discrimination of Influenza A and B

The purpose of this study was to determine the stability of influenza Aand B viruses stored for up to 72 hours at different temperatures indifferent viral transport media as shown by subsequently testing in theapparatus. Saline and eight (8) different commercially available viraltransport media (VTM) were evaluated in this study. One influenza A andone influenza B isolate were used. Five different apparatuses were used.The study spanned a period from zero up to 72 hours. The performance ofthe various VTMs was evaluated at two different controlled temperatures.

The following materials were used in this study: (1) ConsumableMaterials in Table 2-1; (2) Biological Materials in Table 2-2; (3) fivedifferent apparatuses; and, (4) software.

TABLE 2-1 Consumable Materials Description Part Number Lot NumberExpiration Date Influenza A + B Test Cassettes 1169100 2010-1501 TBDReagent Tube 1170700 2010-1267 TBD Pipette, Transfer, 120 uL, 25/Bag1184000 TBD TBD Saline (also Reagent Solution) 0107000 815959 Sep. 30,2013 Media, Micro-Test M5 1187900 954506 Apr. 4, 2012 Media, Micro-TestM4 1189800 029462 Oct. 2, 2012 Media, Universal Viral Transport 118990010A135 Oct. 31, 2012 Media, Micro-Test M4-RT R12505 Remel 015638 Aug.30, 2012 Media, Micro-Test M6 R12530 Remel 951531 Mar. 26, 2012 Media,Hank's Balanced Salts 10-508F 203205 Oct. 18, 2012 BioWhittaker Media,Starplex Multitrans S160-FL Starplex 106H39 September 2012 ModifiedLiquid Stuart's Media SP132-FL Starplex 1C14A Sep. 14, 2012 ControlPositive Swab 1168500 2010-1289 TBD Control Negative Swab 1053300 201613May 8, 2013

TABLE 2-2 Influenza Viruses Virus Strain TCID50/mL A/Hong Kong/8/68 6.1× 106 B/Allen/45 4.2 × 105The five apparatuses used in the study were identified by the followingserial numbers: Analyzer S/N 213; Analyzer S/N 217; Analyzer S/N 219;Analyzer S/N 225; Analyzer S/N 231.Methods

Viral Transport Media are used to stabilize patient specimens when theyare being transported or stored prior to testing. This study examinedthe performance of the test devices and the apparatus when testingsamples diluted and stored in different Viral Transport Media (VTMs) atdifferent temperatures. The study demonstrated the stability of theviruses themselves when stored in different VTMs for up to 72 hours attwo different temperatures.

Some clinics use saline instead of commercial VTMs for transport ortemporary storage of patient specimens. Influenza A and B virusdilutions were therefore prepared in saline. Each dilution contained afinal test concentration 2 to 3 times the LoD for the respectiveviruses. Sufficient volumes were prepared to complete testing at eachtime point. The dilution scheme used for the influenza A and influenza Bvirus is shown in Table 2-3.

Preparation of Virus Dilutions in Viral Transport Media (VTM)

The Influenza A and B virus dilutions were separately prepared invarious types of VTM. Each dilution contained a final test concentration2 to 3 times above the pre-determined LoD. The final reconstitutedextraction reagent contained 260 μL of saline and 260 μL of virusdiluted in different VTMs. Sufficient volumes were prepared to completetesting at each time point. Dilutions of each virus were prepared in thefollowing media: M5, M4, M4-RT, UTM, M6, Hank's Balanced Salts, StarplexMultitrans, and Modified Liquid Stuart's.

TABLE 2-3 Dilution Scheme for Influenza Viruses Virus Virus Vol. Vol.Final Influenza Dilution Dilution Virus Diluent Conc. Vol. Virus # #Used (mL) (mL) (TCID50/mL) (mL) A/Hong 0 NA NA NA 6.11E+06 NA Kong/8/681 0 0.016  4.984 20000  5.000 2 1 0.448 39.552 224 40.000 B/Allen/ 0 NANA NA 4.20E+05 NA 45 1 0 0.095  1.905 20000  2.000 2 1 0.180 39.820 9040.000General Procedure

The Influenza A+B test assay was performed as described in Example 1 andin accord with the package insert. The positive and negative ExternalControls were tested on each day of the study. The apparatus was used inthe Read Now mode throughout the study. All data were stored on a SDcard located in each apparatus; this SD card was removed and the datawere extracted after the testing was completed.

Experimental Protocol

Testing with Saline

Saline without added virus was tested for each condition in replicatesof 5 (n=5); 520 μL of the saline were added into the extraction reagenttube and mixed according to the package insert. This volume was used inorder to mimic the same conditions that are employed when testing withspecimens suspended in VTM as well nasopharyngeal aspirate and washsamples. Next, 120 μL of the reconstituted extraction reagent was addedto the test cassette, using the kit's transfer pipette. The testcassette was incubated for 15 minutes on the benchtop and analyzed usingthe Read Now mode. A new extraction tube was rehydrated for each test.

All virus dilutions prepared in saline were stored at RT or at 2-8° C.and tested in replicates of 5 (n=5) at the following time points: 0 hrs,2 hrs, 4 hrs, 6 hrs, 24 hrs, 48 hrs, and 72 hrs. To test the virusdilutions that had been prepared in saline, 260 μL of the saline wereadded into the extraction reagent tube. Then, 260 μL, of the virusdiluted in saline were added into the extraction reagent tube and mixedaccording to the package insert. Next, 120 μL of the reconstitutedextraction reagent was added to the test cassette, using the kit'stransfer pipette. The test cassette was incubated for 15 minutes on thebenchtop, using the Read Now mode and analyzed. A new extraction tubewas rehydrated for each test.

Testing with Viral Transport Media

The VTMs containing no spiked virus were tested at time zero only. Tenreplicates (n=10) of these unspiked VTM samples were tested. There weretwo temperature conditions evaluated for each medium: RT and 2-8° C. Totest the unspiked VTMs, 260 μL of the saline and 260 μL, of theappropriate VTM were added into the extraction reagent tube and mixed.Next, 120 μL of the reconstituted extraction reagent was added to thetest cassette, using the kit's transfer pipette. The test cassette wasincubated for 15 minutes on the benchtop, using the Read Now mode andanalyzed. A new extraction tube was rehydrated for each test.

All VTMs spiked with virus were tested in replicates of 5 (n=5) at thefollowing time points: 0 hrs, 2 hrs, 24 hrs, 48 hrs, and 72 hrs.Specimens were stored at two different temperature conditions, RT and2-8° C., up to each time point for each of the prepared dilutions. Totest the spiked VTMs, 260 μL of the saline, followed by 260 μL of theappropriate virus dilution in VTM were added into the extraction reagenttube and mixed according to the package insert. Next, 120 μL of thereconstituted extraction reagent were added to the test cassette, usingthe kit's transfer pipette. The test cassette was incubated for 15minutes on the bench top using the Read Now mode and inserted in to theSofia Analyzer for analysis. A new extraction tube was rehydrated foreach test.

Results

The Influenza A and B results were reported as positive, negative orinvalid for each virus dilution and condition. The results for eachvirus at each time point and for each temperature-challenging conditionare presented in Tables 2-4, 2-5, 2-6, and 2-7.

TABLE 2-4 Stability of Influenza A* Stored in Saline and Various VTMs at2-8° C. Number of Positive Results for Influenza A** T = 0 T = 2 T = 4 T= 6 T = 24 T = 48 T = 72 Media hrs hrs hrs hrs hrs hrs hrs Saline 5/55/5 5/5 5/5 5/5 5/5 4/5 M4 5/5 — — 5/5   4/4*** 5/5 5/5 UTM 5/5 — — 5/55/5 5/5 5/5 M5 5/5 — — 5/5 5/5 5/5 5/5 M6 5/5 — — 5/5 5/5 5/5 5/5 M4-RT5/5 — — 5/5 5/5 5/5 5/5 Starplex 5/5 — — 5/5 5/5 5/5 5/5 Hank's 5/5 — —5/5 5/5 3/5 1/5 Stuart's 5/5 — — 5/5 1/5 0/5 0/5 *The influenza A strainused was A/HK/8/68 (TCID50/mL = 224). **There were no false positivesfor influenza B and no invalid results under any condition. ***Onereplicate was mistakenly not tested.

TABLE 2-5 Stability of Influenza A* Stored in Saline and Various VTMs at25° C. Number of Positive Results for Influenza A** T = 0 T = 2 T = 4 T= 6 T = 24 T = 48 T = 72 Media hrs hrs hrs hrs hrs hrs hrs Saline 5/55/5 5/5 4/5 5/5 1/5 0/5 M4 5/5 — — 5/5 5/5 5/5 5/5 UTM 5/5 — — 5/5 5/55/5 5/5 M5 5/5 — — 5/5 5/5 5/5 5/5 M6 5/5 — — 5/5 5/5 5/5 5/5 M4-RT 5/5— — 5/5 5/5 5/5 5/5 Starplex 5/5 — — 5/5 5/5 5/5 5/5 Hank's 5/5 — — 5/54/5 0/5 0/5 Stuart's 5/5 — — 0/5 0/5 0/5 0/5 *The influenza A strainused was A/HK/8/68 (TCID50/mL = 224). **There were no false positivesfor influenza B and no invalid results under any condition.

TABLE 2-6 Stability of Influenza B* Stored in Saline and Various VTMs at2-8° C. Number of Positive Results for Influenza B** T = 0 T = 2 T = 4 T= 6 T = 24 T = 48 T = 72 Media hrs hrs hrs hrs hrs hrs hrs Saline 5/55/5 5/5 5/5 5/5 4/5 0/5 M4 5/5 — — 5/5 5/5 5/5 5/5 UTM 5/5 — — 5/5 5/55/5 5/5 M5 5/5 — — 5/5 5/5 5/5 5/5 M6 5/5 — — 5/5 5/5 5/5 5/5 M4-RT 5/5— — 5/5 5/5 5/5 5/5 Starplex 5/5 — — 5/5 5/5 5/5 5/5 Hank's 5/5 — — 5/55/5 5/5 2/5 Stuart's 5/5 — — 5/5 5/5 0/5 0/5 *The influenza B strainused was B/Allen/45 (TCID50/mL = 90). **There were no false positivesfor influenza A and no invalid results under any condition.

TABLE 2-7 Stability of Influenza B* Stored in Saline and Various VTMs at25° C. Number of Positive Results for Influenza B** T = 0 T = 2 T = 4 T= 6 T = 24 T = 48 T = 72 Media hrs hrs hrs hrs hrs hrs hrs Saline 5/55/5 5/5 5/5 2/5 1/5 0/5 M4 5/5 — — 5/5 5/5 5/5 5/5 UTM 5/5 — — 5/5 5/55/5 5/5 M5 5/5 — — 5/5 5/5 5/5 5/5 M6 5/5 — — 5/5 5/5 5/5 5/5 M4-RT 5/5— — 5/5 5/5 5/5 5/5 Starplex 5/5 — — 5/5 5/5 5/5 5/5 Hank's 5/5 — — 5/54/5 0/5 0/5 Stuart's 5/5 — — 3/5 0/5 0/5 0/5 *The influenza B strainused was B/Allen/45 (TCID50/mL = 90). **There were no false positivesfor influenza A and no invalid results under any condition.

CONCLUSION

Several different VTMs and saline were analyzed for their suitability tostore influenza A and B viruses. Two different temperature conditionswere examined, including ambient RT and 2-8° C. The stability of virusesthat were stored in VTM or saline for 0 hours, 2 hours, 4 hours, 6hours, 24 hours, 48 hours and 72 hours of incubation at 2-8° C. and RTwas examined in the Influenza A+B test device. The study demonstratedthat viruses stored in M4, UTM, M5, M6, M4-RT and Starplex Multitransfor up to 72 hours at either temperature still gave 100% correctresults. Saline was not useable beyond 4 hours at RT, however, it couldbe used for up to 24 hours, if the sample were stored at 2° C. to 8° C.Hank's and Stuart's were inferior and not recommended for storingspecimens at RT. However, Hank's can be used for storing specimens up 24hours and Stuart's up to 6 hours at 2° C. to 8° C. All the media wereshown to be compatible with the Influenza A+B test device; none gavefalse positive or invalid results under the conditions examined. Allstudies were done in full accord with the package insert carefullydescribes the procedure for testing samples suspended in VTM.

Example 3 Fluorescent Immunoassay Showing Improved Sensitivity forDetection of Respiratory Syncytial Virus (RSV

Current Respiratory Syncytial Virus (RSV) rapid tests are visual,subjective tests which claim sensitivity of 83%-95% versus culture.Using the apparatus described herein, instrument-mediated interpretationof the test result eliminates the subjectivity and difficultiessometimes encountered with traditional visually-interpreted, rapidtests. The study described in this example illustrates the improveddetection of RSV using the apparatus. The extraction reagent providedwith a lateral flow immunoassay test kit for detection of RSV wasrehydrated, the nasopharyngeal swab containing a sample was placed intothe extraction reagent and an aliquot of the extracted specimen wastransferred onto the test cassette, which was then placed into theapparatus. All other steps for analysis and interpretation of theresults were performed by the apparatus.

The analytical sensitivity of the RSV detection on the apparatus wascompared to that of the visually-interpreted QuickVue® RSV 10 dipsticktest. Five different strains of RSV (two type A and three type B) werediluted in M5 media to concentrations that yielded an optical density onthe QuickVue visual test that non-technical readers have beendemonstrated to interpret as positive 95% of the time. These dilutionswere considered to be the Limits of Detection (LoD) for the respectivestrains on the QuickVue RSV 10 test. From these concentrations,additional dilutions of 1:100, 1:150, 1:200, 1:300, and 1:400 were madein M5 media for each RSV strain and tested on the RSV immunoassay readon the apparatus. For each viral strain, the highest dilution from theQuickVue LoD that yielded 100% positive results (5/5) was considered tobe the comparative LoD for the RSV immunoassay read on the apparatus.

The clinical specificity of the RSV immunoassay read on the apparatuswas evaluated using 50 nasopharyngeal swab samples that were collectedfrom 50 different asymptomatic donors. To be eligible for the study,donors were required to be free of the common symptoms of RSV infection,including runny nose, congestion, cough, and fever. Swabs were placeddirectly into the rehydrated extraction reagent within one hour ofcollection and tested in the Sofia RSV FIA according to standard packageinsert directions.

Results

In the analytical sensitivity study, the dilutions of each viral strainfrom the QuickVue RSV 10 LoD that yielded 100% positive results on theRSV immunoassay read on the apparatus were 1:200 for strain A-2(TCID₅₀/ml: 1.14×10³); 1:200 for strain A Long (TCID₅₀/ml: 2.87×10³);1:300 for strain B 9320 (TCID₅₀/ml: 3.3×10⁰); 1:100 for strain BWV/14617/85 (TCID₅₀/ml: 3.19×10⁸); and 1:400 for strain B Wash/18537/62(TCID₅₀/ml: 1.1×10¹).

In the clinical specificity study, all 50 of the samples collected fromasymptomatic donors exhibited negative results on the RSV immunoassayread on the apparatus. The RSV immunoassay read on the apparatusdemonstrated 100 to 400 times improved analytical sensitivity comparedto the visually-interpreted dipstick test. A limited clinical study of50 freshly-collected nasopharyngeal swab specimens demonstratedspecificity of 100%.

Example 4 Detection of Group A Streptococcus Via Fluorescent Immunoassay

A clinical study was conducted at six (6) distinct sites in variousgeographical regions within the United States and two (2) sites inAustralia. Two (2) throat swabs were collected from 596 patients withsymptoms suggestive of bacterial pharyngitis. One throat swab wastransported on cold ice packs to a central Reference Laboratory,streaked on a sheep blood agar plate (SBA) and cultured for up to 48hours. Immediately after streaking, this same swab was tested using afluorescent immunoassay lateral flow test strip read in the apparatusdescribed herein. The performance of the system (lateral flowimmunoassay and apparatus) was determined by comparison of the rapidtest result to the corresponding culture result. Bacterial cultures with10 or more GAS-positive colonies in the first quadrant of the streakplate, and zero or more in the other three quadrants were consideredculture-positive.

The other throat swab, collected from the same patient, was testeddirectly in the physician's office or clinic without streaking on SBA.The results were compared to culture obtained with the first swab.

The swab that was sent to the reference laboratory for culture showed aclinical sensitivity of 100% and a specificity of 97% when rare cultureresults (defined by fewer than 10 colonies on a culture plate) wereexcluded. The data set included 79 Group A Streptococcusculture-positive specimens, and 499 culture-negative specimens.

Culture Pos Neg Sofia Pos 79 17 Sofia Neg 0 482 Total: 79 499 Sens =79/79 (100%) (95% C.I. 94-100%) Spec = 482/499 (97%) (95% C.I. 95-98%)PPV = 82% NPV = 100%

Culture Classification of Throat Swab Specimens and Corresponding SofiaStrep A FIA Results Culture Sofia Strep A Classification FIA Result Rare11/18 (61%)  1+  9/9 (100%) 2+ 22/22 (100%) 3+ 29/29 (100%) 4+ 19/19(100%)

There were 18 rarer and six invalids; and these samples were excludedfrom the calculations of clinical accuracy. This clinical data showedcomparable results to traditional culture methods.

Example 5 Comparison of the System with Cell Culture and PCR, forDetection of Influenza A and B

A study was conducted to establish the clinical performance of thesystem described herein, of a lateral flow immunofluorescence assay forinfluenza A and B read using an apparatus. The performance of the systemwas compared to the results obtained using cell culture and PCR.

The lateral flow immunoassay for Influenza A+B FIA test and theMolecular A+B PCR assay were obtained from Quidel Corporation (SanDiego, Calif.). The reference culture method used R-Mix-2™ shell vialssupplied by Diagnostic Hybrids, Inc.

Fresh nasal/nasopharyngeal swab specimens from a total of 929 consentedpatients were included in this analysis. Compared to culture, thefluorescent immunoassay read using the apparatus yielded 96.3%sensitivity and 97.4% specificity for influenza A and 92.3% and 97.9%,respectively for influenza B. Compared to PCR, the fluorescentimmunoassay read using the apparatus yielded 80.7% sensitivity and 99.3%specificity for influenza A and 86.1% and 97.8%, respectively forinfluenza B. The tables below show the performance of the fluorescentimmunoassay read using the apparatus compared to the Ct counts obtainedwith the PCR-positive specimens for influenza A and B. For PCR-specimenswith Ct counts ≤30, fluorescent immunoassay read using the apparatusdetected 96% that were PCR positive for A and 98% that were PCR positivefor B.

The fluorescent immunoassay read using the apparatus yield within 15minutes objective results that demonstrate high clinical accuracy versusculture and PCR, and thus provide ample time at the point-of-care forvalid, informed patient management decisions.

TABLE 5-1 Influenza A Sofia PCR #/N % Ct Positive Positive 95% CI Ct ≤20 32/32 100.0 89.1 to 100  Ct > 20 to ≤25 105/108 97.2 92.1 to 99.4Ct > 25 to ≤30 48/53 90.6 79.3 to 96.9 Ct > 30 to ≤35 10/17 58.8 32.9 to81.6 Ct > 35 to ≤40 2/16 12.5  1.6 to 38.3 Ct > 40 to ≤45 0/18 0  0.0 to18.5

TABLE 5-2 Influenza B Sofia PCR #/N % Ct Positive Positive 95% CI Ct ≤20 5/5 100 47.8 to 100  Ct > 20 to ≤25 68/71 95.8 88.1 to 99.1 Ct > 25to ≤30 71/71 100.0 94.9 to 100  Ct > 30 to ≤35 16/25 64.0 42.5 to 82.0Ct > 35 to ≤40 4/15  26.7  7.8 to 55.1 Ct > 40 to ≤45 3/7 42.9  9.9 to81.6

Example 6 Detection of Influenza A+B

A beta-site study to collect and test prospective clinical specimens wasconducted in January 2011 to further evaluate the test system. Under IRBapproval and informed consent, 98 subjects, all children, contributednasopharyngeal aspirates for testing. One portion of the specimen wastested directly and the other was placed into VTM and then tested,giving a total of 196 test results. The specimen placed into VTM wasalso tested using standard DFA and culture methods; results were furthercompared to an in-house-validated RT-PCR method.

The results compared with culture for Influenza A showed a clinicalsensitivity of 98% and a specificity of 95%, representing 41 Influenza Aculture-positive specimens. For Influenza B, the results showed asensitivity of 100% and a specificity of 99% with a total of 19influenza B positive specimens. Relative to RT-PCR the Influenza Aclinical sensitivity and specificity were 85% and 98%, respectively, andfor Influenza B the clinical sensitivity and specificity were 100% and98%, respectively.

Example 7 Detection of Community Acquired Respiratory Viruses

Twenty influenza viruses were serially diluted in normal saline andtested using the point-of-care apparatus described herein on aninfluenza rapid antigen test strip. The viruses tested consisted of 2H1N1pdm viruses, 1 of each of the 16 HA subtypes of influenza A, and oneof each of the two lineages of influenza B. Logarithmic serial dilutionswere made for each virus and tested in triplicate. Samples were runusing the nasal wash procedure for each assay (i.e. 340 μL of thediluted virus was added directly to the sample extraction reagent).Limits of detection were recorded as the dilution in which at least twoof the three replicates were positive.

The limit of detection for all influenza A virus samples ranged fromranged from 10¹⁸ to 10^(3.55) TCID₅₀/mL in the multiplex influenza rapidantigen test. The multiplex assay showed good dilutional sensitivity forall 20 influenza types and subtypes tested (including both H1N1pdmstrains, the Yamagata lineage influenza B strain, and seven additionalinfluenza A strains).

TABLE 7-1 Quick Vue Fluorescent Flu Fluorescent Flu Influenza A + B A +B Test LOD A + B Test LOD Virus Name Test LOD (MCW Results) (RawResults) A/WI/629-9/2008 (H1N1)  1.0 × 10^(4.55)  1.0 × 10^(4.55)  1.0 ×10^(3.55) A/WI/629-2/2008 (H3N2) 1.0 × 10^(2.8) 1.0 × 10^(2.8) 1.0 ×10^(2.8) A/WI/629-D02473/2009 (H1N1pdm)  1.0 × 10^(3.55)  1.0 ×10^(3.55)  1.0 × 10^(2.55) A/WI/629-D02312/2009 (H1N1pdm) 1.0 × 10^(3.8)1.0 × 10^(3.8) 1.0 × 10^(2.8) A/Mallard/NY/6750/78 (H2N2) 1.0 × 10^(3.8)1.0 × 10^(3.8) 1.0 × 10^(2.8) A/Anhui/01/2005(H5N1)-PR8-IBCDC-RG5  1.0 ×10^(3.05)  1.0 × 10^(2.05)  1.0 × 10^(2.05) A/Chicken/NJ/15086-3/94(H7N3)  1.0 × 10^(3.55)  1.0 × 10^(2.55)  1.0 × 10^(2.55)A/Chicken/NJ/12220/97 (H9N2) 1.0 × 10^(2.8) 1.0 × 10^(2.8) 1.0 ×10^(2.8) B/Ohio/1/2005 (Victoria/2/87-like) 1.0 × 10^(2.8) 1.0 ×10^(2.8) 1.0 × 10^(2.8) B/Florida/07/2004 (Yamagata/16/88-like)  1.0 ×10^(4.05)  1.0 × 10^(4.05)  1.0 × 10^(3.05) A/Mallard/OH/338/86 (H4N8) 1.0 × 10^(3.55)  1.0 × 10^(3.55)  1.0 × 10^(3.55) A/Chicken/CA/431/00(H6N2) 1.0 × 10^(3.8) 1.0 × 10^(3.8) 1.0 × 10^(2.8) A/Blue WingedTeal/LA/B194/86 (H8N4) 1.0 × 10^(2.8) 1.0 × 10^(2.8) 1.0 × 10^(2.8)A/GWT/LA/169GW/88 (H10N7)  1.0 × 10^(3.05)  1.0 × 10^(3.05)A/Chicken/NJ/15902-9/96 (H11N9)  1.0 × 10^(3.55)  1.0 × 10^(4.55)  1.0 ×10^(3.55) A/Duck/LA/188D/87 (H12N5)  1.0 × 10^(4.05)  1.0 × 10^(4.05) 1.0 × 10^(3.05) A/Gull/MD/704/77 (H13N6) 1.0 × 10^(1.8) 1.0 × 10^(1.8)1.0 × 10^(1.8) A/Mallard/GurjevRussia/262/82 (H14N5) 1.0 × 10^(1.8) 1.0× 10^(1.8) 1.0 × 10^(1.8) A/Shearwater/Australia/2576/79 (H15N9) 1.0 ×10^(2.8) 1.0 × 10^(2.8) 1.0 × 10^(2.8) A/Shorebird/DE/172/2006(H16N3)1.0 × 10^(2.8) 1.0 × 10^(1.8) 1.0 × 10^(1.8)

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

It is claimed:
 1. An apparatus, comprising: a housing configured toreceive a test device, wherein the test device comprises a referenceline and an analyte-specific test line; an illumination source; adetector; and a processor comprising a software program for control ofthe illumination source and the detector and for processing dataobtained from the detector; wherein the processor is configured todirect the apparatus to: (a) generate a one-dimensional data array ofvalues corresponding to signal associated with the reference line andthe analyte-specific test line in response to illumination by theillumination source, (b) use data in the data array corresponding todata from the reference line to identify a location of data in the dataarray that corresponds to the analyte-specific test line, and (c)analyze data in the data array that corresponds to the analyte-specifictest line to report a result regarding presence or absence of an analyteat the analyte-specific test line.
 2. The apparatus of claim 1, whereinthe detector is a single-element photodetector.
 3. The apparatus ofclaim 1, wherein the illumination source is a light emitting diode. 4.The apparatus of claim 3, wherein the light emitting diode emits lightat about 365 nm.
 5. The apparatus of claim 3, further comprising a poweroutput that provides the light emitting diode with at least about 4 mW.6. The apparatus of claim 1, further comprising a cellular componentthat provides wireless transmission of data.
 7. The apparatus of claim6, wherein the cellular component transmits the result obtained fromanalysis of the data in the data array.
 8. The apparatus of claim 1,wherein the apparatus further comprises a port for connection with anexternal instrument.
 9. The apparatus of claim 1, wherein the processoris configured to direct the apparatus to analyze data in the data arrayby calculating a first derivative of the data array to identify aminimum peak and a maximum peak corresponding to the reference line. 10.The apparatus of claim 1, wherein the processor is configured to directthe apparatus to analyze data in the data array by taking the firstderivative of the data array to form a derivative data set, wherein afirst maximal value in the derivative data set corresponds to a maximumsignal from a reference line or a control line on the test device, andthe position in the derivate data array of the first maximal valuedetermines the position of data from the analyte-specific test line. 11.The apparatus of claim 1, wherein the detector detects signal emittedfrom the test device during illumination by the illumination source. 12.The apparatus of claim 11, wherein the emitted signal is emitted from afluorescing lanthanide compound.
 13. The apparatus of claim 12, whereinthe fluorescing lanthanide compound is chelated europium.
 14. Theapparatus of claim 13, wherein the chelated europium is embedded inmicroparticles.